ATP Regulation of Type 1 Inositol 1,4,5-Trisphosphate Receptor Channel Gating by Allosteric Tuning of Ca2+ Activation
1999; Elsevier BV; Volume: 274; Issue: 32 Linguagem: Inglês
10.1074/jbc.274.32.22231
ISSN1083-351X
AutoresDon‐On Daniel Mak, Sean McBride, J. Kevin Foskett,
Tópico(s)Pancreatic function and diabetes
ResumoInositol 1,4,5-trisphosphate (InsP3) mobilizes intracellular Ca2+ by binding to its receptor (InsP3R), an endoplasmic reticulum-localized Ca2+ release channel. Patch clamp electrophysiology of Xenopus oocyte nuclei was used to study the effects of cytoplasmic ATP concentration on the cytoplasmic Ca2+ ([Ca2+]i) dependence of single type 1 InsP3R channels in native endoplasmic reticulum membrane. Cytoplasmic ATP free-acid ([ATP]i), but not the MgATP complex, activated gating of the InsP3-liganded InsP3R, by stabilizing open channel state(s) and destabilizing the closed state(s). Activation was associated with a reduction of the half-maximal activating [Ca2+]ifrom 500 ± 50 nm in 0 [ATP]i to 29 ± 4 nm in 9.5 mm [ATP]i, with apparent ATP affinity = 0.27 ± 0.04 mm, similar to in vivo concentrations. In contrast, ATP was without effect on maximum open probability or the Hill coefficient for Ca2+activation. Thus, ATP enhances gating of the InsP3R by allosteric regulation of the Ca2+ sensitivity of the Ca2+ activation sites of the channel. By regulating the Ca2+-induced Ca2+ release properties of the InsP3R, ATP may play an important role in shaping cytoplasmic Ca2+ signals, possibly linking cell metabolic state to important Ca2+-dependent processes. Inositol 1,4,5-trisphosphate (InsP3) mobilizes intracellular Ca2+ by binding to its receptor (InsP3R), an endoplasmic reticulum-localized Ca2+ release channel. Patch clamp electrophysiology of Xenopus oocyte nuclei was used to study the effects of cytoplasmic ATP concentration on the cytoplasmic Ca2+ ([Ca2+]i) dependence of single type 1 InsP3R channels in native endoplasmic reticulum membrane. Cytoplasmic ATP free-acid ([ATP]i), but not the MgATP complex, activated gating of the InsP3-liganded InsP3R, by stabilizing open channel state(s) and destabilizing the closed state(s). Activation was associated with a reduction of the half-maximal activating [Ca2+]ifrom 500 ± 50 nm in 0 [ATP]i to 29 ± 4 nm in 9.5 mm [ATP]i, with apparent ATP affinity = 0.27 ± 0.04 mm, similar to in vivo concentrations. In contrast, ATP was without effect on maximum open probability or the Hill coefficient for Ca2+activation. Thus, ATP enhances gating of the InsP3R by allosteric regulation of the Ca2+ sensitivity of the Ca2+ activation sites of the channel. By regulating the Ca2+-induced Ca2+ release properties of the InsP3R, ATP may play an important role in shaping cytoplasmic Ca2+ signals, possibly linking cell metabolic state to important Ca2+-dependent processes. Modulation of free cytoplasmic Ca2+ concentration ([Ca2+]i) is a ubiquitous cellular signaling system. In many cell types, binding of ligands to plasma membrane receptors activates the hydrolysis of phosphatidylinositol 4,5-bisphosphate by membrane-bound phospholipase C, generating inositol 1,4,5-trisphosphate (InsP3). 1The abbreviations used are: InsP3, inositol 1,4,5-trisphosphate; InsP3R, InsP3receptor; ER, endoplasmic reticulum; BAPTA, 1,2-bis(O-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid; LTD, long term depression; mGluR, metabotropic glutamate receptor(s) InsP3 causes the release of Ca2+ from the endoplasmic reticulum (ER) by binding to its receptor (InsP3R), which itself is a Ca2+ channel (1Taylor C.W. Richardson A. Pharmacol. Ther. 1991; 51: 97-137Crossref PubMed Scopus (122) Google Scholar, 2Berridge M.J. Nature. 1993; 361: 315-325Crossref PubMed Scopus (6187) Google Scholar, 3Putney Jr., J.W. St J. Bird G. Endocr. Rev. 1993; 14: 610-631Crossref PubMed Scopus (486) Google Scholar). 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Acta. 1998; 1366: 17-32Crossref PubMed Scopus (95) Google Scholar). [Ca2+]i signals generated by InsP3R-mediated Ca2+ release from the ER appear to be rapidly and efficiently transmitted to mitochondria (28Rizzuto R. Brini M. Murgia M. Pozzan T. Science. 1993; 262: 744-747Crossref PubMed Scopus (1016) Google Scholar, 29Rizzuto R. Bastianutto C. Brini M. Murgia M. Pozzan T. J. Cell Biol. 1994; 126: 1183-1194Crossref PubMed Scopus (309) Google Scholar, 30Csordás G. Thomas A.P. Hajnóczky G. EMBO J. 1999; 18: 96-108Crossref PubMed Scopus (454) Google Scholar), acutely affecting mitochondrial functions (31McCormack J.G. Denton R.M. Dev. Neurosci. 1993; 15: 165-173Crossref PubMed Scopus (130) Google Scholar, 32Loew L.M. Carrington W. Tuft R.A. Fay F.S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12579-12583Crossref PubMed Scopus (122) Google Scholar, 33Rutter G.A. Burnett P. Rizzuto R. Brini M. Murgia M. Pozzan T. Tavaré J.M. Denton R.M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5489-5494Crossref PubMed Scopus (221) Google Scholar), including ATP synthesis (34Robb-Gaspers L.D. Burnett P. Rutter G.A. Denton R.M. Rizzuto R. Thomas A.P. EMBO J. 1998; 17: 4987-5000Crossref PubMed Scopus (323) Google Scholar). It is unknown, however, whether the communication between these two organelles is reciprocal. ATP release from mitochondria, globally into the cytoplasm and locally into the vicinity of the InsP3R channels that are in close apposition, may provide a signaling pathway for communication from the mitochondria back to the ER. Thus, regulation of the InsP3R by ATP could have considerable significance for intracellular signaling, particularly if the channel is sensitive to ATP levels in normal physiological as well as pathological conditions, including ischemia. Most previous studies of ATP regulation of the InsP3R have been limited to indirect measurements, i.e. Ca2+fluxes or concentrations, to infer InsP3R channel activity, because the intracellular location of the Ca2+ release channel has limited its accessibility to electrophysiological approaches. Furthermore, only a limited range of [Ca2+]i was examined in previous studies, despite the fact that the InsP3R is intricately regulated by [Ca2+]i (6Finch E.A. Turner T.J. Goldin S.M. Science. 1991; 252: 443-446Crossref PubMed Scopus (676) Google Scholar, 8Mak D.-O.D. McBride S. Foskett J.K. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15821-15825Crossref PubMed Scopus (230) Google Scholar, 9Iino M. J. Gen. Physiol. 1990; 95: 1103-1122Crossref PubMed Scopus (496) Google Scholar, 10Marshall I.C.B. Taylor C.W. J. Biol. Chem. 1993; 268: 13214-13220Abstract Full Text PDF PubMed Google Scholar, 11Bezprozvanny I. Watras J. Ehrlich B.E. Nature. 1991; 351: 751-754Crossref PubMed Scopus (1441) Google Scholar) and that the primary known regulator of the channel, InsP3, mediates its effects by modulating the [Ca2+]i dependence of channel gating (8Mak D.-O.D. McBride S. Foskett J.K. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15821-15825Crossref PubMed Scopus (230) Google Scholar). Therefore, in the present study, we have systematically investigated the effects of cytoplasmic ATP concentration on the [Ca2+]i response of single InsP3R channels. We applied the patch clamp technique to isolatedXenopus oocyte nuclei (35Mak D.-O.D. Foskett J.K. J. Biol. Chem. 1994; 269: 29375-29378Abstract Full Text PDF PubMed Google Scholar, 36Mak D.-O.D. Foskett J.K. J. Gen. Physiol. 1997; 109: 571-587Crossref PubMed Scopus (142) Google Scholar, 37Stehno-Bittel L. Lückhoff A. Clapham D.E. Neuron. 1995; 14: 163-167Abstract Full Text PDF PubMed Scopus (176) Google Scholar) to study the single channel activities of the type 1 InsP3R (InsP3R-1), the major brain isoform (38Furuichi T. Yoshikawa S. Miyawaki A. Wada K. Maeda N. Mikoshiba K. Nature. 1989; 342: 32-38Crossref PubMed Scopus (826) Google Scholar, 39Mignery G.A. Newton C.L. Archer III, B.T. Sudhof T.C. J. Biol. Chem. 1990; 265: 12679-12685Abstract Full Text PDF PubMed Google Scholar), in its native ER membrane environment under rigorously defined conditions on both the cytoplasmic and luminal sides of the channel. Our results demonstrate that cytoplasmic ATP free acid, but not cytoplasmic MgATP complex, activates the gating of the InsP3R primarily by allosteric regulation of the [Ca2+]i sensitivity of the Ca2+activation sites of the channel. By regulating the Ca2+-induced Ca2+ release properties of the InsP3R, ATP may play an important role in shaping the extent and duration of [Ca2+]i signals, possibly linking cell metabolic state to important Ca2+-dependent process including synaptic plasticity. Patch clamp experiments were performed as described in Refs. 8Mak D.-O.D. McBride S. Foskett J.K. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15821-15825Crossref PubMed Scopus (230) Google Scholar, 35Mak D.-O.D. Foskett J.K. J. Biol. Chem. 1994; 269: 29375-29378Abstract Full Text PDF PubMed Google Scholar, 36Mak D.-O.D. Foskett J.K. J. Gen. Physiol. 1997; 109: 571-587Crossref PubMed Scopus (142) Google Scholar, and 40Mak D.-O.D. Foskett J.K. Am. J. Physiol. 1998; 275: C179-C188Crossref PubMed Google Scholar. Briefly, stage V or VI oocytes were opened mechanically just prior to use. The nucleus was separated from the cytoplasm and transferred to a dish on the stage of a microscope for patch-clamping. The oocyte expresses only a single InsP3R isoform (type 1) and lacks other (e.g.ryanodine receptor) Ca2+ release channels (41Kume S. Muto A. Aruga J. Nakagawa T. Michikawa T. Furuichi T. Nakade S. Okano H. Mikoshiba K. Cell. 1993; 73: 555-570Abstract Full Text PDF PubMed Scopus (204) Google Scholar). Experiments were done in "on-nucleus" configuration, with the solution in the perinuclear lumen between the outer and inner nuclear membranes in apparent equilibrium with the bath solution (35Mak D.-O.D. Foskett J.K. J. Biol. Chem. 1994; 269: 29375-29378Abstract Full Text PDF PubMed Google Scholar) and with the cytoplasmic aspect of the InsP3R channel facing into the patch pipette. Following standard conventions, the applied potential is that of the pipette electrode minus the reference bath electrode (positive current flows from pipette outward). Experiments were performed at room temperature with the pipette electrode at +20 mV relative to the reference bath electrode. Single channel currents were amplified with an Axopatch-1D amplifier (Axon Instruments, Foster City, CA) with antialiasing filtering at 1 kHz, transferred to a Power Macintosh 8100 via an ITC-16 interface (Instrutech Corp., Great Lake, NY), digitized at 5 kHz, and written directly onto the hard disc by Pulse+PulseFit software (HEKA Elektronik, Lambrecht, Germany). Data were analyzed to identify channel opening and closing events and evaluate channel open probabilities using MacTac 3 (Bruxton, Seattle, WA). Each data point shown is the mean of results from at least four separate patch clamp experiments performed under the same conditions. Error bars indicate the S.E. Theoretical curves were fitted to experimental data using Igor Pro 3 (WaveMetrics, Lake Oswego, OR). All patch clamp experiments were performed with solutions containing 140 mmKCl and 10 mm HEPES with pH adjusted to 7.1 using KOH. Since the luminal [Ca2+] or [ATP] have no systematic effects on the open probability response of the InsP3R (8Mak D.-O.D. McBride S. Foskett J.K. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15821-15825Crossref PubMed Scopus (230) Google Scholar), a bath solution containing no ATP and 250 nm free Ca2+ was used in all experiments. Pipette solutions contained various concentrations of nucleotides (sodium salts of ATP, ADP, AMP, GTP, and UTP and adenosine, from Sigma) as specified. Because of chelation of Mg2+ by ATP, the actual free [Mg2+] and free [ATP] in the solutions containing Mg2+ and ATP were calculated by the Maxchelator software (C. Patton, Stanford University, Stanford, CA). By using K+as the current carrier and appropriate quantities of the high affinity Ca2+ chelator, BAPTA (100–500 μm; Molecular Probes, Inc., Eugene, OR), the low affinity Ca2+ chelator, 5,5′-dibromo-BAPTA (100–350 μm; Molecular Probes), or just ATP (0 or 0.5 mm) to buffer [Ca2+] in the experimental solutions, [Ca2+] was tightly controlled in our experiments. Total Ca2+ content (60–370 μm) in the solutions was determined by induction-coupled plasma mass spectrometry (Mayo Medical Laboratory, Rochester, MN). Free [Ca2+] was calculated using the Maxchelator software. Pipette solutions contained 10 μm InsP3(Molecular Probes). To examine the effects of ATP on the permeation and gating properties of the InsP3R, we included 10 μm InsP3, 250 nmfree Ca2+, and 0.5 mm free ATP in the pipette solution. Under these conditions, the endogenous Xenopustype 1 InsP3R channel exhibited channel conductance properties and kinetics similar to those observed previously under similar conditions (8Mak D.-O.D. McBride S. Foskett J.K. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15821-15825Crossref PubMed Scopus (230) Google Scholar, 40Mak D.-O.D. Foskett J.K. Am. J. Physiol. 1998; 275: C179-C188Crossref PubMed Google Scholar). The channels gated with a moderately high open probability (P o) of ∼0.5 (Fig.1 A). In similar experiments employing pipette solutions that lacked ATP, the InsP3R channel P o was significantly lower (∼0.2) in either the absence or presence of 3 mm Mg2+(Fig. 1, B and C). To determine whether the ATP activation of the channel P o was mediated by MgATP, which could suggest a role for ATP hydrolyisis or phosphorylation, similar experiments were undertaken with 3 mm total Mg2+ and 0.5 mm total ATP in the pipette. Under these conditions, [MgATP] is approximately 0.5 mm, and the free Mg2+ concentration ([Mg2+]i) and the cytoplasmic free ATP concentration ([ATP]i) were calculated to be 2.5 and 0.012 mm, respectively. Nevertheless, P oremained low (Fig. 1 D). The low P o in the presence of MgATP (Fig. 1) was solely caused by ATP complexation by Mg2+, since it was fully reversed by adding more ATP to the pipette solution to restore [ATP]i (Fig. 1 E). Thus, MgATP has no effect, stimulatory or inhibitory, on InsP3R activity. We previously demonstrated that theP o of the Xenopus type 1 InsP3R is independent of [Mg2+]i up to 9.5 mm (40Mak D.-O.D. Foskett J.K. Am. J. Physiol. 1998; 275: C179-C188Crossref PubMed Google Scholar). Taken together, these results suggest that ATP free acid (ATP3− or ATP4−) was the relevant ionic species and that ATP hydrolysis was not involved in the stimulation of InsP3R channel gating. InsP3 activates the InsP3R by modulating the sensitivity of the channel to [Ca2+]i (8Mak D.-O.D. McBride S. Foskett J.K. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15821-15825Crossref PubMed Scopus (230) Google Scholar). To determine the mechanism of ATP activation of the InsP3R channel gating, we investigated in detail the effects of ATP on the channel kinetics of the InsP3R over a wide range of [Ca2+]i. A systematic series of patch clamp experiments were performed using pipette solutions containing various [Ca2+]i with 0.5 mm ATP alone, 3 mm Mg2+ alone, 0.5 mm ATP, and 3 mm Mg2+(calculated [ATP]i = 0.012 mm; calculated [Mg2+]i = 2.5 mm) or no ATP or Mg2+. To avoid possible effects of Ca2+ on InsP3 binding, a functionally saturating InsP3concentration of 10 μm was used (8Mak D.-O.D. McBride S. Foskett J.K. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15821-15825Crossref PubMed Scopus (230) Google Scholar). The [Ca2+]i sensitivity of the InsP3R in the absence of cytoplasmic free ATP was biphasic (Fig.2) and could be well fitted with a biphasic Hill equation similar to the following one previously derived for the InsP3R in the presence of 0.5 mmcytoplasmic free ATP (8Mak D.-O.D. McBride S. Foskett J.K. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15821-15825Crossref PubMed Scopus (230) Google Scholar). Po=Pmax{1+(Kact/[Ca2+]i)Hact}−1{1+([Ca2+]i/Kinh)Hinh}−1.Equation 1 Similar results were obtained independent of the presence or absence of either Mg2+ or MgATP in the pipette solutions. This result indicates that the InsP3R can achieve a maximum open probability P max of 0.79 in the absence of cytoplasmic free ATP, a level of activity very similar to theP max of 0.81 found in the presence of 0.5 mm cytoplasmic free ATP (8Mak D.-O.D. McBride S. Foskett J.K. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15821-15825Crossref PubMed Scopus (230) Google Scholar). Thus, ATP does not activate the channel by increasing P max. The Hill coefficient for Ca2+ activation H actwas 2.4 ± 0.6 in the absence of free ATP, similar toH act = 1.9 ± 0.3 in the presence of 0.5 mm free ATP. This result suggests that Ca2+probably activates the InsP3R via the same cooperative process in either the presence or absence of cytoplasmic free ATP. Thus, ATP does not activate the channel by modulatingH act. The observed activation of the InsP3R by cytoplasmic free ATP (Fig. 1) was associated with a reduction of the half-maximal activating [Ca2+]i (K act) from 500 ± 50 nm in the absence of free ATP to 190 ± 20 nm in the presence of 0.5 mm free ATP. Thus, ATP activates the channel by sensitizing it to Ca2+. ATP therefore enhances Ca2+-induced Ca2+ release (CICR) by the InsP3R. Because InsP3 activates channel gating by modifying the [Ca2+]i inhibition phase of the channel [Ca2+]i dependence (8Mak D.-O.D. McBride S. Foskett J.K. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15821-15825Crossref PubMed Scopus (230) Google Scholar), we were also interested in examining the effects of ATP on this aspect of the response. However, investigations of InsP3R channel activity in the absence of free ATP at [Ca2+]i which inhibit channel gating (>20 μm; Ref. 8Mak D.-O.D. McBride S. Foskett J.K. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15821-15825Crossref PubMed Scopus (230) Google Scholar) were not possible because of the unavailability of a Ca2+ chelator with the appropriate Ca2+ affinity. In our previous experiments that examined the effects of high [Ca2+]i on InsP3R channel gating (8Mak D.-O.D. McBride S. Foskett J.K. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15821-15825Crossref PubMed Scopus (230) Google Scholar), ATP was used as the Ca2+ chelator for buffering [Ca2+]iat high [Ca2+]i. The data we were able to obtain in the absence of ATP in the present study indicated thatP o began to decrease as [Ca2+]i was increased beyond 10 μm, but the inhibitory half-maximal [Ca2+]i,K inh, or Hill coefficient,H inh, could not be determined accurately (Fig.2). Analysis of the mean open and closed durations of the InsP3R revealed that the mean open duration (τo) in the absence of free ATP lay within a narrow range between 5 and 15 ms over a wide range of [Ca2+]i(1–10 μm). At both very low ( 10 μm) [Ca2+]i, τo was shorter (∼3 ms) (Fig.3). In contrast, the mean closed duration (τc) in the absence of free ATP decreased about 2 orders of magnitude, from 200 to 3 ms, as [Ca2+]i was increased from 200 nm to 1 μm. τc remained low between 1 and 10 μm[Ca2+]i (Fig. 4). These same basic kinetics were observed in all experiments conducted in the absence of cytoplasmic free ATP, regardless of the presence or absence of free Mg2+ or MgATP complex. Similar [Ca2+]i dependences of τo and τc (Figs. 3 and 4) were also observed in the presence of 0.5 mm free ATP (8Mak D.-O.D. McBride S. Foskett J.K. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15821-15825Crossref PubMed Scopus (230) Google Scholar). An examination of the differences in the [Ca2+]i dependences of τo and τc in either the presence and absence of cytoplasmic free ATP reveals that the mechanism whereby free ATP enhances channel activity is by stabilization of the open channel state(s) and destabilization of the closed channel state(s) in the low [Ca2+]i regime (30–500 nm). Our more limited data indicate that free ATP may also stabilize the open channel state(s) at very high [Ca2+]i (>15 μm).Figure 4[Ca2+]i dependence of the mean closed durations of the InsP3R channels in the absence of free cytoplasmic ATP (A) and in the presence of 0.5 mm free ATP (B). Thesymbols used are the same as those in Fig. 2.View Large Image Figure ViewerDownload (PPT) We undertook a systematic study of the activation of the InsP3R by [Ca2+]iover a wide range of free [ATP]i. In the presence of 10 μm InsP3, the activating Hill equation (Equation 2) agreed well the experimental data for [ATP]i of 4.8 and 9.5 mm, with no significant effects of [ATP]i on H act orP max (Fig. 5). Po=Pmax{1+(Kact/[Ca2+]i)Hact}−1Equation 2 [ATP]i decreased K act of the InsP3R by over an order of magnitude, from 500 ± 50 nm in 0 mm [ATP]i to 29 ± 4 nm in 9.5 mm [ATP]i. The effects of [ATP]i on K act of the InsP3R were analyzed by fitting the data with a modified Michaelis-Menten equation (Fig.6). Kact=Kmin+Kr{1+([ATP]i/KATP)}−1Equation 3 A modification to the standard Michaelis-Menten equation was necessary because ATP alone is insufficient to activate the channel in absence of InsP3, and therefore the activating half-maximal [Ca2+]i (K act) should not approach 0 even in presence of saturating concentrations of ATP. The results indicate that the range over which K actof the InsP3R varies in response to [ATP]i,K r = 480 ± 20 nm; the minimumK act under saturating [ATP]i,K min = 17 ± 3 nm; and the functional dissociation coefficient for cytoplasmic free ATP activation of the InsP3R, K ATP = 0.27 ± 0.04 mm. The good fit of this equation to the data suggests that ATP stimulation of channel activity is not cooperative, requiring binding of only one ATP molecule to the InsP3R tetramer to stimulate it.Figure 6Effect of [ATP]i on the activating half-maximal [Ca2+]i(K act) of the InsP3R . Thecurve is the theoretical fit based on the modified Michaelis-Menten equation (Equation 3).View Large Image Figure ViewerDownload (PPT) To determine the nucleotide specificity of the stimulatory effects we observed for free ATP, we also investigated the effects of adenosine, AMP, ADP, GTP, and UTP. Each nucleotide was present as 0.5 mm free nucleotide in the absence of Mg2+. The [Ca2+]i was fixed at 220 ± 15 nm, because theP o is very sensitive to activation by free ATP at this [Ca2+]i (Fig. 2). Using the channelP o (0.14) in the absence of any nucleotide as the reference, the relative P o of the channel was determined in the presence of the various nucleotide species (Fig.7). Similar to the MgATP complex, free UTP, ADP, and adenosine had no effects on the P oof the InsP3R (p > 0.05). In contrast, free ATP, AMP, and GTP each activated the InsP3R (p < 0.05). Free ATP had the greatest effect, more than tripling the P o, whereas both free AMP and GTP doubled the channel P o. We previously described the detailed permeation and gating properties of the Xenopus type 1 InsP3R channel by patch clamp studies of isolated oocyte nuclei (8Mak D.-O.D. McBride S. Foskett J.K. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15821-15825Crossref PubMed Scopus (230) Google Scholar, 35Mak D.-O.D. Foskett J.K. J. Biol. Chem. 1994; 269: 29375-29378Abstract Full Text PDF PubMed Google Scholar, 36Mak D.-O.D. Foskett J.K. J. Gen. Physiol. 1997; 109: 571-587Crossref PubMed Scopus (142) Google Scholar, 40Mak D.-O.D. Foskett J.K. Am. J. Physiol. 1998; 275: C179-C188Crossref PubMed Google Scholar). Under optimal conditions, gating of the channel is robust, with maximum open probability of ∼80% over a wide range of [Ca2+]i (8Mak D.-O.D. McBride S. Foskett J.K. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15821-15825Crossref PubMed Scopus (230) Google Scholar). Importantly, the gating of the channel is regulated by both InsP3 as well as by [Ca2+]i. The regulation of the InsP3-liganded channel activity by [Ca2+]i is biphasic, with half-maximal activation at 210 nm and half-maximal inhibition at 45 μm in 10 μm InsP3. InsP3 binds noncooperatively to a high affinity site (K D ∼ 50 nm) on each monomer of the channel tetramer. Binding of InsP3 to the channel has the sole effect of decreasing the Ca2+ affinity of the Ca2+ inhibition site on each monomer, in a process which has high cooperativity (Hill coefficient of 4) (8Mak D.-O.D. McBride S. Foskett J.K. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15821-15825Crossref PubMed Scopus (230) Google Scholar). When cytoplasmic InsP3 concentration ([InsP3]i) is low, under conditions of no or weak stimulation, the channel is inhibited by relatively low [Ca2+]i, whereas it becomes much less sensitive to Ca2+ inhibition at higher [InsP3]i, enabling it to become activated. Thus, InsP3 activates the channel by tuning the inhibition efficacy of the Ca2+ ligand. Of particular significance, [Ca2+]i activation of the channel wasnot modified by InsP3. However, it is unknown whether other modulators of InsP3R activity similarly impinge on the Ca2+ inhibition properties of the channel or whether the Ca2+ activation properties of the channel are exploited as an alternate method of channel regulation. The results from the present study suggest that ATP stimulates gating of the InsP3R by modulating the Ca2+ sensitivity of the Ca2+ activation sites. We performed a systematic investigation of the effects of nucleotides on gating of the Xenopus type 1 InsP3R Ca2+ release channel. Our study focused on the effects of ATP on single channel activity, and included additional examination of effects of other nucleotides for comparison. Stimulation of InsP3R by nucleotides in the presence of InsP3 has been previously reported (20Ferris C.D. Huganir R.L. Snyder S.H. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2147-2151Crossref PubMed
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